ABSTRACT The immune system is one of our best guards against developing cancer and when this system fails cancer is allowed to grow and thrive. Over the last several decades it has become possible to isolate a patient's own immune cells, expand and reactivate them in the laboratory, and use them to fight an existing cancer, termed cancer immunotherapy. This has been accomplished with great success using T cells in the form of tumor infiltrating lymphocytes (TILs) and T Cell Receptor (TCR)/Chimeric Antigen Receptor (CAR)-T cell therapy. However, there are drawbacks to these technologies and they have largely only demonstrated efficacy for blood cancers in the clinic, with the exception of TIL based therapy for melanoma and renal cell carcinoma. One of the drawbacks of TCR/CAR-T based cell therapies is the need for a priori knowledge of tumor specific antigens to be effective. A more ideal approach would use a cell type that naturally targets transformed cells without the need for tumor specific antigens, such as Natural Killer (NK) cells. In fact, there is a surging interest in the use of NK cells for cancer immunotherapy for just this reason, however, the use of unmanipulated NK cells for these therapies has showed some but minimal efficacy; with the exception of some rare instances, such as KIR mismatch. For instance, members of our team have performed clinical trials and generated preclinical models of ovarian cancer that are partially responsive to NK cell immunotherapy. These preclinical ovarian models are ideal for testing enhanced NK treatments as they are partially responsive to standard NK therapy but are unable to eradicate tumors, in part due to inherent NK and tumor mediated NK cell inhibition. NK function is regulated at many levels to avoid autoimmunity and it has been demonstrated that tumor cells take advantage of these systems to avoid being eliminated by NK cells. This is likely one of the main limiting factors in treating cancer with NK cell based immunotherapies. In order to overcome this issue, and enhance NK cells ability to kill cancer cells, we will utilize CRISPR/Cas9 to knockout regulatory genes of NK cell function. We have generated preliminary data demonstrating successful gene editing of human NK cells using CRISPR/Cas9 that result in advantageous functional changes. Therefore, our project objectives are to optimize our ability to genetically modify specific regulatory genes of NK cells (specifically, PD1, ADAM17, and CISH), expand them to clinically relevant numbers, and assess for enhancement in their ability kill ovarian cancer cells in vitro and in vivo. We strongly believe NK immunotherapy can be fully realized by using genome engineering to enhance the anti-cancer function of primary human NK cells. Moreover, since NK cell mediated cytotoxicity does not rely on the presentation of self human leukocyte antigens (HLA) they are highly suited for use in allogeneic settings and therefore hold significant clinical potential as an off-the-shelf cellular product.